Anti-ADDL monoclonal antibody and use thereof

ABSTRACT

The present invention relates to antibodies that differentially recognize multi-dimensional conformations of Aβ-derived diffusible ligands, also known as ADDLs. The antibodies of the invention can distinguish between Alzheimer&#39;s Disease and control human brain extracts and are useful in methods of detecting ADDLs and diagnosing Alzheimer&#39;s Disease. The present antibodies also block binding of ADDLs to neurons, assembly of ADDLs, and tau phosphorylation and are there useful in methods for the preventing and treating diseases associated with soluble oligomers of amyloid β 1-42.

INTRODUCTION

This application is a continuation of U.S. patent application Ser. No.11/581,843 filed Oct. 17, 2006 now U.S. Pat. No. 7,731,962, which is acontinuation-in-part of U.S. patent application Ser. No. 11/256,332,filed Oct. 21, 2005 now U.S. Pat No. 7,780,963, which claims the benefitof priority from U.S. provisional patent application Ser. No.60/652,538, filed Feb. 14, 2005, whose contents are incorporated hereinby reference in their entireties.

BACKGROUND OF THE INVENTION

Alzheimer's Disease is a progressive and degenerative dementia (Terry,et al. (1991) Ann. Neurol. 30:572-580; Coyle (1987) In: Encyclopedia ofNeuroscience, Adelman (ed.), Birkhäuser, Boston-Basel-Stuttgart, pp29-31). In its early stages, Alzheimer's Disease manifests primarily asa profound inability to form new memories (Selkoe (2002) Science298:789-791), reportedly due to neurotoxins derived from amyloid beta(Aβ). Aβ is an amphipathic peptide whose abundance is increased bymutations and risk factors linked to Alzheimer's Disease. Fibrils formedfrom Aβ constitute the core of amyloid plaques, which are hallmarks ofan Alzheimer's Disease brain. Analogous fibrils generated in vitro arelethal to cultured brain neurons. These findings indicate that memoryloss is a consequence of neuron death caused by fibrillar Aβ.

Despite strong experimental support for fibrillar Aβ and memory loss, apoor correlation exists between dementia and amyloid plaque burden(Katzman (1988) Ann. Neurol. 23:138-144). Moreover, transgenic hAPP mice(Dodart, et al. (2002) Nat. Neurosci. 5:452-457; Kotilinek, et al.(2002) J. Neurosci. 22:6331-6335), which develop age-dependent amyloidplaques and, most importantly, age-dependent memory dysfunction, showthat within 24 hours of vaccination with monoclonal antibodies againstAβ memory loss can be reversed with no change in plaque levels. Suchfindings are not consistent with a mechanism for memory loss dependenton neuron death caused by amyloid fibrils.

Additional neurologically active molecules formed by Aβ self-assemblyhave been suggested. These molecules include soluble Aβ oligomers, alsoreferred to as Aβ-derived diffusible ligands or ADDLs. Oligomers aremetastable and form at low concentrations of Aβ1-42 (Lambert, et al.(1998) Proc. Natl. Acad. Sci. USA 95:6448-6453). Aβ oligomers rapidlyinhibit long-term potentiation (LTP), a classic experimental paradigmfor memory and synaptic plasticity. As such, memory loss stems fromsynapse failure, prior to neuron death and synapse failure by Aβoligomers, not fibrils (Hardy & Selkoe (2002) Science 297:353-356).Soluble oligomers have been found in brain tissue and are strikinglyelevated in Alzheimer's Disease (Kayed, et al. (2003) Science300:486-489; Gong, et al. (2003) Proc. Natl. Acad. Sci. USA100:10417-10422) and in hAPP transgenic mice Alzheimer's Disease models(Kotilinek, et al. (2002) J. Neurosci. 22:6331-6335; Chang, et al.(2003) J. Mol. Neurosci. 20:305-313).

A variety of Alzheimer's Disease treatment options have been suggested.Vaccine clinical trials have revealed that persons mounting a vigorousimmune response to the vaccine exhibit cognitive benefit (Hock, et al.(2003) Neuron 38:547-554); however, frequency of CNS inflammation causedearly termination of part of the trial (Birmingham & Frantz (2002)Nat.Med. 8:199-200). As an alternative to a vaccine, therapeutic antibodiesthat target ADDLs without binding monomers or fibrils have beensuggested (Klein (2002) Neurochem. Int. 41:345-352). ADDLs are highlyantigenic, generating oligomer-selective polyclonal antibodies inrabbits at concentration of ˜50 μg/mL (Lambert, et al. (2001) J.Neurochem. 79:595-605). Results from transgenic mice models also suggestthat antibodies can be successful in reversing memory decline (Dodart,et al. (2002) Nat. Neurosci. 5:452-457; U.S. patent application Ser. No.11/194,989). Accordingly, there is a need in the art for ADDL-selectivetherapeutic antibodies for the prevention and treatment of Alzheimer'sDisease. The present invention meets this need.

SUMMARY OF THE INVENTION

The present invention is an isolated antibody, or fragment thereof,capable of differentially recognizing a multi-dimensional conformationof one or more Aβ-derived diffusible ligands. In particular, theantibody of the instant invention has a complementary determining region(CDR) of Arg-Xaa₁-Leu-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Asp-Ala-Met-Asp-Tyr (SEQID NO:9), wherein Xaa₁ is Gln or Ala; Xaa₂ is Ser or Gly; Xaa₃ is Pro,Ala, Lys, Arg, or Thr; Xaa₄ is Lys or Arg; Xaa₅ is Gly, Ser, or Lys;Xaa₆ is Val, Thr, Ile or Arg. In particular embodiments, the antibody ofthe present invention is in admixture with a pharmaceutically acceptablecarrier. In other embodiments, the antibody of the present invention isin a kit. Still other embodiments embrace an antibody having heavy andlight chain variable region sequences as set forth in SEQ ID NO:108 andSEQ ID NO:112. An antibody having heavy and light chain sequences as setforth in SEQ ID NO:138 and SEQ ID NO:140 is also provided.

Methods for preventing binding of Aβ-derived diffusible ligands to aneuron and inhibiting assembly of Aβ-derived diffusible ligandsemploying an antibody or antibody fragment which binds amulti-dimensional conformation of one or more Aβ-derived diffusibleligands are also provided.

The present invention further embraces a method for prophylactically ortherapeutically treating a disease associated with Aβ-derived diffusibleligands using an antibody of the instant invention. Administration of anantibody of the invention can prevent binding of Aβ-derived diffusibleligands to a neuron thereby preventing or treating the diseaseassociated with Aβ-derived diffusible ligands.

The present invention is also a method for identifying a therapeuticagent that prevents the binding of Aβ-derived diffusible ligands to aneuron. This method of the invention involves contacting a neuron withAβ-derived diffusible ligands in the presence of an agent and using anantibody of the present invention to determine binding of the Aβ-deriveddiffusible ligands to the neuron in the presence of the agent.

The present invention also embraces a method for detecting Aβ-deriveddiffusible ligands in a sample and a method for diagnosing a diseaseassociated with Aβ-derived diffusible ligands. Such methods involvecontacting a sample with an antibody of the instant invention so thatthe Aβ-derived diffusible ligands can be detected and a diseaseassociated with Aβ-derived diffusible ligands can be diagnosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleic acid sequences for the heavy (FIG. 1A) andlight (FIG. 1B) chain variable regions for murine anti-ADDL antibody20C2. Lower case letters indicate the antibody leader sequences anduppercase letters indicate antibody variable region sequences. Thenucleotides coding for the complementary determining regions (CDRs) areunderlined.

FIG. 2 shows comparisons of heavy (FIG. 2A) and light (FIG. 2B) chainvariable region amino acid sequences of murine antibody 20C2 andhumanized antibodies, Hu20C2 (CDR grafted) and Hu20C2A3 (veneered).Sequences are presented as comparisons between the 20C2 mouse sequence,the most homologous human genomic sequence and the humanized sequences.Sequence differences in the frame regions between murine 20C2 andhumanized Hu20C2A3 are in bold. Sequence differences in the underlinedCDR regions between humanized Hu20C2A3 and murine 20C2 are in bold andindicated with an *. CDRs are underlined.

FIG. 3 shows nucleic acid sequences for the heavy (FIGS. 3A and 3B) andlight (FIG. 3C) chain variable regions (HCVRs and LCVRs, respectively)for humanized anti-ADDL antibody Hu20C2 (CDR grafted). Two humanizedversions of the Hu20C2 heavy chain were generated (HCVRA and HCVRB) thatdiffer by one amino acid at position 24. In Hu20C2 HCVRA the human aminoacid was used and in Hu20C2 HCVRB the mouse amino acid was used.Variable region sequences were cloned into full heavy and light chainantibody expression vectors.

FIG. 4 shows the annotated amino acid sequences and nucleotide sequencesof Hu20C2 humanized antibody in Fab phage-display vector pFab4. Aminoacid sequence for heavy chain version A (FIG. 4A), heavy chain version B(FIG. 4B), and the light chain (FIG. 4C) of Hu20C2 humanized antibody inFab phage-display vector pFab4 are in italic and underlined regions areas indicated. Nucleotide sequence of heavy chain version A fused withthe light chain of Hu20C2 in pFab4 vector is shown in FIG. 4D-4E withsequences encoding the Hu20C2 antibody sequences shown in lowercase.

FIG. 5 depicts the design and primers employed in preparing two lightchain CDR3 libraries, namely LC3-1 and LC3-2 (FIG. 5A), and three heavychain CDR3 libraries, namely 20C2B-39HC₃-1, 20C2B-39HC₃-2, and20C2B-39HC₃-3 (FIG. 5B), for respectively generating affinity maturedHu20C2 light and heavy chain CDR3s. Restriction endonuclease recognitionsites used for cloning are indicated in italic. Uppercase indicatesnucleic acids encoding antibody variable region sequences. Nucleic acidsencoding CDRs are underlined. Biotin-labeled primers are indicated.

FIG. 6 shows a comparison of the amino acid sequence of human antibodyconstant regions and the sequence of IgG2m4. The asterisk indicates aglycosylation site at Asn297. Regions of FcRn binding are indicated.Sequences in which IgG2m4 is different from IgG2 are underlined.

FIG. 7 shows the amino acid (FIGS. 7A and 7C) and nucleotide (FIGS. 7Band 7D) sequences for the full IgG2m4 humanized heavy chain (FIGS. 7Aand 7B) and humanized Kappa light chain (FIGS. 7C and 7D) for anti-ADDLantibody Hu20C2A3. Underlining indicates variable region sequences. Theremaining sequences are constant region sequences.

FIG. 8 shows interactions between Aβ40 monomer or ADDLs with Hu20C2A3produced by two different systems, CHO (FIG. 8A) or Pichia (FIG. 8B), asdetermined by ELISA.

FIG. 9 shows Hu20C2A3 inhibition of bADDL binding to primary hippocampalneurons.

FIG. 10 shows fluorescent thermal melt analysis of Hu20C2A3.

FIG. 11 shows plasma Aβx-40 levels (pM) of APP-YAC mice followingintravenous injection of Hu20C2A3, irrelevant control or vehicle.Hu20C2A3 was prepared by stable transfection of CHO cells or Pichia.Aβx-40 was determined using a 4G8/G2-10 ELISA. ***, p<0.001 byTukey-Kramer HSD post-hoc testing. Error bars=SEM; N=6/group.

DETAILED DESCRIPTION OF THE INVENTION

Monoclonal antibodies, which differentially recognize multi-dimensionalconformations of Aβ-derived diffusible ligands (i.e., ADDLs), have nowbeen generated. Antibodies of this invention are derived from the murinemonoclonal antibody 20C2. Murine 20C2 is known in the art for exhibitingthe following characteristics. Murine 20C2 is an IgG1 antibody whichbinds to both synthetic and endogenous ADDLs bound to culturedhippocampal cells. Furthermore, this antibody can block both endogenousand synthetic ADDL binding to cultured cells, abate the binding ofbiotinylated ADDLs (bADDLs) to neurons, and prevent tau phosphorylation.The core linear epitope for 20C2 is Glu-Phe-Arg-His-Asp-Ser (SEQ IDNO:1), corresponding to amino acid residues 3-8 of Aβ1-42, with aconformational epitope that is dependent upon elements from withinresidues 17-42 of Aβ, but only when assembled.

The instant antibodies are humanized and, in some embodimentsaffinity-matured derivatives of murine 20C2. Like the murine 20C2antibody, the antibodies disclosed herein exhibit a high degree ofselectivity for multi-dimensional conformations of ADDLs, with minimaldetection of monomer Aβ peptides. Advantageously, the instant antibodiesidentify endogenous oligomers in Alzheimer's Disease brain slices andinhibit binding of bADDLs to neurons. Moreover, the instant antibodiesprovide a significant and robust increase in plasma Aβx-40 levels, anincrease in which is known to be associated with an ultimate lowering ofbrain Aβ. Accordingly, the antibodies of this invention find use in theprevention of ADDL binding to neurons and assembly of ADDLs and thetreatment of ADDL-related diseases including Alzheimer's Disease.

Accordingly, the present invention is an isolated antibody thatdifferentially recognizes one or more multi-dimensional conformations ofADDLs. An antibody of the instant invention is said to be isolated whenit is present in the substantial absence of other biologicalmacromolecules of the same type. Thus, an “isolated antibody” refers toan antibody which is substantially free of other antibodies; however,the molecule may include some additional agents or moieties which do notdeleteriously affect the basic characteristics of the antibody (e.g.,binding specificity, neutralizing activity, etc.).

An antibody which is capable of specifically binding one or moremulti-dimensional conformations of ADDLs, binds particular ADDLs derivedfrom the oligomerization of Aβ1-42, but like murine 20C2 does notcross-react with other Aβ peptides, namely Aβ1-12, Aβ1-28, Aβ1-40, andAβ12-28 as determined by western blot analyses; and preferentially bindADDLs in solution. Specific binding between two entities generallyrefers to an affinity of at least 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹.Affinities greater than 10⁸ M⁻¹ are desired to achieve specific binding.

In particular embodiments, an antibody that is capable of specificallybinding a multi-dimensional conformation of one or more ADDLs is alsoraised against (i.e., an animal is immunized with) multi-dimensionalconformations of ADDLs. In other embodiments, an antibody that iscapable of specifically binding a multi-dimensional conformation of oneor more ADDLs is raised against a low n-mer-forming peptide such asAβ-42[Nle35-Dpro37].

The term “epitope” refers to a site on an antigen to which B and/or Tcells respond or a site on a molecule against which an antibody will beproduced and/or to which an antibody will bind. For example, an epitopecan be recognized by an antibody defining the epitope.

A linear epitope is an epitope wherein an amino acid primary sequencecomprises the epitope recognized. A linear epitope typically includes atleast 3, and more usually, at least 5, for example, about 6 to about 10amino acids in a unique sequence.

A conformational epitope, in contrast to a linear epitope, is an epitopewherein the primary sequence of the amino acids comprising the epitopeis not the sole defining component of the epitope recognized (e.g., anepitope wherein the primary sequence of amino acids is not necessarilyrecognized by the antibody defining the epitope). Typically aconformational epitope encompasses an increased number of amino acidsrelative to a linear epitope. With regard to recognition ofconformational epitopes, the antibody recognizes a three-dimensionalstructure of the peptide or protein. For example, when a proteinmolecule folds to form a three-dimensional structure, certain aminoacids and/or the polypeptide backbone forming the conformational epitopebecome juxtaposed enabling the antibody to recognize the epitope.Methods of determining conformation of epitopes include but are notlimited to, for example, x-ray crystallography, two-dimensional nuclearmagnetic resonance spectroscopy and site-directed spin labeling andelectron paramagnetic resonance spectroscopy. See, for example, EpitopeMapping Protocols in Methods in Molecular Biology (1996) Vol. 66, Morris(Ed.).

Aβ-derived diffusible ligands or ADDLs refer to soluble oligomers ofamyloid β1-42 which are desirably composed of aggregates of less thaneight or nine amyloid β1-42 peptides and are found associated withAlzheimer's Disease. This is in contrast to high molecular weightaggregation intermediates, which form stings of micelles leading tofibril formation.

As exemplified herein, the instant antibody binds or recognizes at leastone multi-dimensional conformation of an ADDL. In particularembodiments, the instant antibody binds at least two, at least three, orat least four multi-dimensional conformations of an ADDL.Multi-dimensional conformations of ADDLs are intended to encompassdimers, trimers, tetramers pentamers, hexamers, heptamers, octamers,nonamers, decamers, etc. as defined by analysis via SDS-PAGE. Becausetrimer, tetramer, etc. designations can vary with the assay methodemployed (see, e.g., Bitan, et al. (2005) Amyloid 12:88-95) thedefinition of trimer, tetramer, and the like, as used herein, isaccording to SDS-PAGE analysis. As such, the antibody of the instantinvention has oligomer-specific characteristics. In particularembodiments, a multi-dimensional conformation of an ADDL is associatedwith a specific polypeptide structure which results in a conformationalepitope that is recognized by an antibody of the present invention. Inother embodiments, an antibody of the invention specifically binds amulti-dimensional conformation ADDL having a size range of approximatelya trimer or tetramer, which have molecular weights in excess of >50 kDa.

In certain embodiments, in addition to binding to a multi-dimensionalconformation, the instant antibody binds to a selected linear epitope ofamyloid β1-42. A linear epitope of an ADDLs is intended as a four, five,six or more amino acid residue peptide located in the N-terminal 10, 11,12, 15 or 20 amino acid residues of amyloid β1-42. In particularembodiments, an antibody of the invention specifically binds to a linearepitope within residues 1-10, 1-8, 3-10, or 3-8 of amyloid β1-42. Anexemplary linear epitope of amyloid β1-42 which is bound by a humanizedantibody of the invention is amino acid residues Glu-Phe-Arg-His-Asp-Ser(SEQ ID NO:1).

While antibodies of the instant invention may have similar linearepitopes, such linear epitopes are not wholly indicative of the bindingcharacteristics of the instant antibodies (i.e., ability to block ADDLbinding to neurons, prevent tau phosphorylation and inhibit ADDLassembly) because, as is well-known to the skilled artisan, the linearepitope may only correspond to a portion of the antigen's epitope (see,e.g., Breitling and Dübel (1999) In: Recombinant Antibodies, John Wiley& Sons, Inc., NY, pg. 115). For example, murine 20C2 is known to bindassemblies of charge-inverted, truncated Aβ7-42 peptide, which lack thelinear epitope for 20C2 (i.e., amino acid residues 3-8) and contain avery different sequence corresponding to residues 7-16 of Aβ. Therefore,20C2, as well as humanized derivatives thereof, bind to conformationalepitopes that depend upon elements from within residues 17-42 of Aβ, butonly when in a multidimensional conformation. The antibody of theinstant invention can be distinguished from those of the art as beingcapable of differentially recognizing multi-dimensional ADDLs andaccordingly differentially blocking ADDL binding to neurons,differentially preventing tau phosphorylation and differentiallyinhibiting ADDL assembly.

An antibody, as used in accordance with the instant invention includes,but is not be limited to, monoclonal antibodies, and chimeric, human(e.g. isolated from B cells), humanized, neutralizing, bispecific orsingle chain antibodies thereof. In one embodiment, an antibody of theinstant invention is monoclonal. For the production of antibodies,various hosts including goats, rabbits, chickens, rats, mice, humans,and others, can be immunized by injection with synthetic or naturalADDLs. Methods for producing antibodies are well-known in the art. See,e.g., Kohler and Milstein ((1975) Nature 256:495-497) and Harlow andLane (Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory,New York (1988)).

Depending on the host species, various adjuvants can be used to increasethe immunological response. Adjuvants used in accordance with theinstant invention desirably augment the intrinsic response to ADDLswithout causing conformational changes in the immunogen that affect thequalitative form of the response. Particularly suitable adjuvantsinclude 3 De-O-acylated monophosphoryl lipid A (MPL™; RIBI ImmunoChemResearch Inc., Hamilton, Mont.; see GB 2220211) and oil-in-wateremulsions, such as squalene or peanut oil, optionally in combinationwith immune stimulants, such as monophosphoryl lipid A (see Stoute, etal. (1997) N. Engl. J. Med. 336:86-91), muramyl peptides (e.g.,N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(E-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP)), or other bacterial cell wall components.Specific examples of oil-in-water emulsions include MF59 (WO 90/14837),containing 5% Squalene, 0.5% TWEEN™ 80, and 0.5% SPAN 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton, Mass.); SAF containing 10% Squalene, 0.4% TWEEN™80, 5% PLURONIC®-blocked polymer L121, and thr-MDP, eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion; and RIBI™ adjuvant system (RAS) (RibiImmunoChem, Hamilton, Mont.) containing 2% squalene, 0.2% TWEEN™ 80, andone or more bacterial cell wall components such as monophosphoryllipidA, trehalose dimycolate (TDM), and cell wall skeleton (CWS).

Another class of adjuvants is saponin adjuvants, including ISCOMs(immunostimulating complexes) and ISCOMATRIX® (CSL Ltd., Parkville,Australia). Other suitable adjuvants include Complete Freund's Adjuvant(CFA), Incomplete Freund's Adjuvant (IFA), mineral gels such as aluminumhydroxide, and surface-active substances such as lysolecithin, PLURONIC®polyols, polyanions, peptides, CpG (WO 98/40100), keyhole limpethemocyanin, dinitrophenol, and cytokines such as interleukins (IL-1,IL-2, and IL-12), macrophage colony stimulating factor (M-CSF), andtumor necrosis factor (TNF). Among adjuvants used in humans, BCG(bacilli Calmette-Guerin) and Corynebacterium parvum are particularlysuitable.

An antibody to a multi-dimensional conformation ADDL is generated byimmunizing an animal with ADDLs. Generally, ADDLs can be generatedsynthetically or by recombinant fragment expression and purification.Synthetic ADDLs can be prepared as disclosed herein or in accordancewith the methods disclosed in U.S. Pat. No. 6,218,506 or in co-pendingapplications U.S. Ser. Nos. 60/621,776, 60/652,538, 60/695,528 and60/695,526. Further, ADDLs can be fused with another protein such askeyhole limpet hemocyanin to generate an antibody against the chimericmolecule. The ADDLs can be conformationally constrained to form anepitope useful as described herein and furthermore can be associatedwith a surface for example, physically attached or chemically bonded toa surface in such a manner so as to allow for the production of aconformation which is recognized by the antibodies of the presentinvention.

Monoclonal antibodies to multi-dimensional conformations of ADDLs can beprepared using any technique which provides for the production ofantibody molecules by continuous cell lines in culture. These include,but are not limited to, the hybridoma technique, the human B-cellhybridoma technique, and the EBV-hybridoma technique (Kohler, et al.(1975) Nature 256:495-497; Kozbor, et al. (1985) J. Immunol. Methods81:31-42; Cote, et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole,et al. (1984) Mol. Cell. Biol. 62:109-120).

In particular embodiments, the instant antibodies are humanized.Humanized or chimeric antibodies can be produced by splicing of mouseantibody genes to human antibody genes to obtain a molecule withappropriate antigen specificity and biological activity (see Morrison,et al. (1984) Proc. Natl. Acad. Sci. 81, 6851-6855; Neuberger, et al.(1984) Nature 312:604-608; Takeda, et al. (1985) Nature 314:452-454;Queen, et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033; WO90/07861). For example, a mouse antibody is expressed as the Fv or Fabfragment in a phage selection vector. The gene for the light chain (andin a parallel experiment, the gene for the heavy chain) is exchanged fora library of human antibody genes. Phage antibodies, which still bindthe antigen, are then identified. This method, commonly known as chainshuffling, provided humanized antibodies that should bind the sameepitope as the mouse antibody from which it descends (Jespers, et al.(1994) Biotechnology NY 12:899-903). As an alternative, chain shufflingcan be performed at the protein level (see, Figini, et al. (1994) J.Mol. Biol. 239:68-78).

Human antibodies can also be obtained using phage-display methods. See,e.g., WO 91/17271 and WO 92/01047. In these methods, libraries of phageare produced in which members display different antibodies on theirouter surfaces. Antibodies are usually displayed as Fv or Fab fragments.Phage displaying antibodies with a desired specificity are selected byaffinity enrichment to ADDLs. Human antibodies against ADDLs can also beproduced from non-human transgenic mammals having transgenes encoding atleast a segment of the human immunoglobulin locus and an inactivatedendogenous immunoglobulin locus. See, e.g., WO 93/12227 and WO 91/10741.Human antibodies can be selected by competitive binding experiments, orotherwise, to have the same epitope specificity as a particular mouseantibody. Such antibodies generally retain the useful functionalproperties of the mouse antibodies. Human polyclonal antibodies can alsobe provided in the form of serum from humans immunized with animmunogenic agent. Optionally, such polyclonal antibodies can beconcentrated by affinity purification using ADDLs as an affinityreagent.

As exemplified herein, humanized antibodies can also be produced byveneering or resurfacing of murine antibodies. Veneering involvesreplacing only the surface fixed region amino acids in the mouse heavyand light variable regions with those of a homologous human antibodysequence. Replacing mouse surface amino acids with human residues in thesame position from a homologous human sequence has been shown to reducethe immunogenicity of the mouse antibody while preserving its ligandbinding. The replacement of exterior residues generally has little, orno, effect on the interior domains, or on the interdomain contacts.(See, e.g., U.S. Pat. No. 6,797,492).

Human or humanized antibodies can be designed to have IgG, IgD, IgA, IgMor IgE constant regions, and any isotype, including IgG1, IgG2, IgG3 andIgG4. In particular embodiments, an antibody of the invention is IgG orIgM, or a combination thereof. Other embodiments of the presentinvention embrace a constant region formed by selective incorporation ofhuman IgG4 sequences into a standard human IgG2 constant region. Anexemplary mutant IgG2 Fc is IgG2m4, set forth herein as SEQ ID NO:140.Antibodies can be expressed as tetramers containing two light and twoheavy chains, as separate heavy chains and light chains or as singlechain antibodies in which heavy and light chain variable domains arelinked through a spacer. Techniques for the production of single chainantibodies are well-known in the art.

Exemplary humanized antibodies derivatives of murine 20C2 monoclonalantibody are provided herein by CDR grafting and veneering. Amino acidsequences for IgG2M4 heavy chain variable regions, as well as kappalight chain variable regions for humanized 20C2 (i.e., Hu20C2A3)generated by veneering are presented in FIGS. 7A and 7C and set forthherein as SEQ ID NO:141 and SEQ ID NO:143.

Diabodies are also contemplated. A diabody refers to an engineeredantibody construct prepared by isolating the binding domains (both heavyand light chain) of a binding antibody, and supplying a linking moietywhich joins or operably links the heavy and light chains on the samepolypeptide chain thereby preserving the binding function (see, Holligeret al. (1993) Proc. Natl. Acad. Sci. USA 90:6444; Poljak (1994)Structure 2:1121-1123). This forms, in essence, a radically abbreviatedantibody, having only the variable domain necessary for binding theantigen. By using a linker that is too short to allow pairing betweenthe two domains on the same chain, the domains are forced to pair withthe complementary domains of another chain and create twoantigen-binding sites. These dimeric antibody fragments, or diabodies,are bivalent and bispecific. The skilled artisan will appreciate thatany method to generate diabodies can be used. Suitable methods aredescribed by Holliger, et al. (1993) supra, Poljak (1994) supra, Zhu, etal. (1996) Biotechnology 14:192-196, and U.S. Pat. No. 6,492,123,incorporated herein by reference.

Fragments of an isolated antibody of the invention are also expresslyencompassed by the instant invention. Fragments are intended to includeFab fragments, F(ab′)₂ fragments, F(ab′) fragments, bispecific scFvfragments, Fd fragments and fragments produced by a Fab expressionlibrary, as well as peptide aptamers. For example, F(ab′)₂ fragments areproduced by pepsin digestion of the antibody molecule of the invention,whereas Fab fragments are generated by reducing the disulfide bridges ofthe F(ab′)₂ fragments. Alternatively, Fab expression libraries can beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity (see Huse, et al. (1989) Science254:1275-1281). In particular embodiments, antibody fragments of thepresent invention are fragments of neutralizing antibodies which retainthe variable region binding site thereof. Exemplary are F(ab′)₂fragments, F(ab′) fragments, and Fab fragments. See generallyImmunology: Basic Processes (1985) 2^(nd) edition, J. Bellanti (Ed.) pp.95-97.

Peptide aptamers which differentially recognize multi-dimensionalconformations of ADDLs can be rationally designed or screened for in alibrary of aptamers (e.g., provided by Aptanomics SA, Lyon, France). Ingeneral, peptide aptamers are synthetic recognition molecules whosedesign is based on the structure of antibodies. Peptide aptamers consistof a variable peptide loop attached at both ends to a protein scaffold.This double structural constraint greatly increases the binding affinityof the peptide aptamer to levels comparable to that of an antibody(nanomolar range).

Exemplary nucleic acid sequences encoding heavy and light chain variableregions for use in producing antibody and antibody fragments of theinstant invention are respectively disclosed herein in FIGS. 7B and 7D(i.e., SEQ ID NOs:142 and 144). As will be appreciated by the skilledartisan, the heavy chain variable regions disclosed herein can be usedin combination with any one of the light chain variable regionsdisclosed herein to generate antibodies with modified affinities,dissociate constants, epitopes and the like.

Antibodies or antibody fragments of the present invention can haveadditional moieties attached thereto. For example, a microsphere ormicroparticle can be attached to the antibody or antibody fragment, asdescribed in U.S. Pat. No. 4,493,825, the disclosure of which isincorporated herein by reference.

Moreover, particular embodiment embrace antibody or antibody fragmentswhich are mutated and selected for increased antigen affinity,neutralizing activity (i.e., the ability to block binding of ADDLs toneuronal cells or the ability to block ADDL assembly), or a modifieddissociation constant. Mutator strains of E. coli (Low, et al. (1996) J.Mol. Biol. 260:359-368), chain shuffling (Figini, et al. (1994) supra),and PCR mutagenesis are established methods for mutating nucleic acidmolecules encoding antibodies. By way of illustration, increasedaffinity can be selected for by contacting a large number of phageantibodies with a low amount of biotinylated antigen so that theantibodies compete for binding. In this case, the number of antigenmolecules should exceed the number of phage antibodies, but theconcentration of antigen should be somewhat below the dissociationconstant. Thus, predominantly mutated phage antibodies with increasedaffinity bind to the biotinylated antigen, while the larger part of theweaker affinity phage antibodies remains unbound. Streptavidin can thenassist in the enrichment of the higher affinity, mutated phageantibodies from the mixture (Schier, et al. (1996) J. Mol. Biol.255:28-43). Exemplary affinity-maturated light chain CDR3 amino acidsequences are disclosed herein (see Tables 6 and 7), with particularembodiments embracing a light chain CDR3 amino acid sequence ofXaa₁-Gln-Xaa₂-Thr-Arg-Val-Pro-Leu-Thr (SEQ ID NO:2), wherein Xaa₁ is Pheor Leu, and Xaa₂ is Ala or Thr. Affinity-maturated heavy chain CDR3amino acid sequences are also provided herein. An exemplary heavy chainCDR3 amino acid sequence is set forth herein as Arg-Gln-Leu-Gly-Thr-Arg-Gly-Thr-Asp-Ala-Met-Asp-Tyr (SEQ ID NO:3). Thepresent invention also embraces derivatives of this CDR3, e.g.,Arg-Ala-Leu-Ser-Pro-Arg-Ser-Ile-Asp-Ala -Met-Asp-Tyr (SEQ ID NO:4),Arg-Gln-Leu-Gly-Ala-Arg-Lys-Thr -Asp-Ala-Met-Asp-Tyr (SEQ ID NO:5),Arg-Gln-Leu-Gly-Pro-Arg -Lys-Arg-Asp-Ala-Met-Asp-Tyr (SEQ ID NO:6),Arg-Gln-Leu-Gly -Lys-Leu-Lys-Thr-Asp-Ala-Met-Asp-Tyr (SEQ ID NO:7), orArg -Gln-Leu-Gly-Arg-Arg-Ser-Val-Asp-Ala-Met-Asp-Tyr (SEQ ID NO:8),wherein differences with the Hu20C2A3 heavy chain CDR3 are underlined.In this regard, the present invention specifically embraces an anti-ADDLantibody having a CDR3 amino acid sequence ofArg-Xaa₁-Leu-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Asp-Ala-Met-Asp-Tyr (SEQ ID NO:9),wherein Xaa₁ is Gln or Ala; Xaa₂ is Ser or Gly; Xaa₃ is Pro, Ala, Lys,Arg, or Thr; Xaa₄ is Lys or Arg; Xaa₅ is Gly, Ser, or Lys; Xaa₆ is Val,Thr, Ile or Arg

Other antibody derivatives encompassed within the scope of the presentinvention include any humanized antibody identical to Hu20C2A3'svariable regions except with a one amino acid residue difference in theframe region of the light chain (e.g., Leu-Pro-Val-Thr-Pro-Gly-Glu-Pro-Ala-Ser, SEQ ID NO:10).

For some therapeutic applications it may be desirable to reduce thedissociation of the antibody from the antigen. To achieve this, phageantibodies are bound to biotinylated antigen and an excess ofunbiotinylated antigen is added. After a period of time, predominantlythe phage antibodies with the lower dissociation constant can beharvested with streptavidin (Hawkins, et al. (1992) J. Mol. Biol.226:889-96).

Various immunoassays including those disclosed herein can be used forscreening to identify antibodies, or fragments thereof, having thedesired specificity for multi-dimensional conformations of ADDLs.Numerous protocols for competitive binding (e.g, ELISA), latexagglutination assays, immunoradiometric assays, kinetics (e.g., BIACORE™analysis) using either polyclonal or monoclonal antibodies, or fragmentsthereof, are well-known in the art. Such immunoassays typically involvethe measurement of complex formation between a specific antibody and itscognate antigen. A two-site, monoclonal-based immunoassay utilizingmonoclonal antibodies reactive to two non-interfering epitopes issuitable, but a competitive binding assay can also be employed. Suchassays can also be used in the detection of multi-dimensionalconformations of ADDLs in a sample.

An antibody or antibody fragment can also be subjected to otherbiological activity assays, e.g., displacement of ADDL binding toneurons or cultured hippocampal cells or blockade of ADDL assembly, inorder to evaluate neutralizing or pharmacological activity and potentialefficacy as a prophylactic or therapeutic agent. Such assays aredescribed herein and are well-known in the art.

Antibodies and fragments of antibodies can be produced and maintained ashydridomas or alternatively recombinantly produced in anywell-established expression system including, but not limited to, E.coli, yeast (e.g., Saccharomyces spp. and Pichia spp.), baculovirus,mammalian cells (e.g., myeloma, CHO, COS), plants, or transgenic animals(Breitling and Dübel (1999) In: Recombinant Antibodies, John Wiley &Sons, Inc., NY, pp. 119-132). Antibodies and fragments of antibodies canbe isolated using any appropriate methods including, but not limited to,affinity chromatography, immunoglobulins-binding molecules (e.g.,proteins A, L, G or H), tags operatively linked to the antibody orantibody fragment (e.g., His-tag, FLAG®-tag, Strep tag, c-myc tag) andthe like. See, Breitling and Dübel (1999) supra.

Antibodies and antibody fragments of the instant invention have avariety of uses including, diagnosis of diseases associated withaccumulation of ADDLs, blocking or inhibiting binding of ADDLs toneuronal cells, blocking ADDL assembly, prophylactically ortherapeutically treating a disease associated with ADDLs, identifyingtherapeutic agents that prevent binding of ADDLs to neurons, andpreventing the phosphorylation of tau protein at Ser202/Thr205.

Antibody and antibody fragments of the instant invention are also usefulin a method for blocking or inhibiting binding of ADDLs to neuronalcells. This method of the invention is carried out by contacting aneuron, in vitro or in vivo, with an antibody or antibody fragment ofthe present invention so that binding of ADDLs to the neuron is blocked.In particular embodiments, an antibody or antibody fragment of theinstant invention achieves at least a 15%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or 97% decrease in the binding of ADDLs as compared tobinding of ADDLs in the absence of the antibody or antibody fragment.The degree to which an antibody can block the binding of ADDLs to aneuron can be determined in accordance with the methods disclosedherein, i.e., immunocytochemistry or cell-based alkaline phosphataseassay or any other suitable assay. Antibodies particularly useful fordecreasing binding of ADDLs to neuronal cells include anti-ADDLantibodies having a CDR3 amino acid sequence set forth in SEQ ID NO:9,as well as derivatives and fragments thereof.

Antibody and antibody fragments of the instant invention are furtheruseful in a method for blocking or inhibiting assembly of ADDLs. Thismethod involves contacting a sample containing amyloid β 1-42 peptideswith an antibody or antibody fragment of the instant invention so thatADDL assembly is inhibited. The degree to which an antibody can blockthe assembly of ADDLs can be determined in accordance with the methodsdisclosed herein, i.e., FRET or fluorescence polarization or any othersuitable assay. Antibodies particularly useful for blocking the assemblyof ADDLs include anti-ADDL antibodies having a CDR3 amino acid sequenceset forth in SEQ ID NO:9, as well as derivatives and fragments thereof.

Antibodies disclosed herein are also useful in methods for preventingthe phosphorylation of tau protein at Ser202/Thr205. This methodinvolves contacting a sample containing tau protein with an antibody orantibody fragment of the instant invention so that binding of ADDLs toneurons is blocked thereby preventing phosphorylation of tau protein.The degree to which an antibody can prevent the phosphorylation of tauprotein at Ser202/Thr205 can be determined in accordance with themethods disclosed herein or any other suitable assay.

Blocking or decreasing binding of ADDLs to neurons, inhibiting assemblyof ADDLs, and preventing the phosphorylation of tau protein atSer202/Thr205 all find application in methods of prophylactically ortherapeutically treating a disease associated with the accumulation ofADDLs. Accordingly, the present invention also embraces the use of anantibody or antibody fragment of the instant invention to prevent ortreat a disease associated with the accumulation of ADDLs (e.g.Alzheimer's or similar memory-related disorders). Evidence in the artindicates that elevated levels of Aβ, but not necessarily aggregatedplaque, are causative for Alzheimer's Disease-associated dementia andsubsequent tau abnormalities. Aβ-derived diffusible ligands are directlyimplicated in neurotoxicity associated with Alzheimer's Disease. The artindicates that ADDLs are elevated in transgenic mice and Alzheimer'sDisease patients and modulate functional activity associated withmnemonic processes in animal models. Thus, removing this form of Aβcould provide relief from the neurotoxicity associated with Alzheimer'sDisease. As such, treatment with the instant antibody, which reducescentral nervous system ADDL load, could prove efficacious for thetreatment of Alzheimer's Disease. Patients amenable to treatment includeindividuals at risk of disease but not exhibiting symptoms, as well aspatients presently exhibiting symptoms. In the case of Alzheimer'sDisease, virtually anyone is at risk of suffering from Alzheimer'sDisease if he or she lives long enough. Therefore, the antibody orantibody fragments of the present invention can be administeredprophylactically to the general population without the need for anyassessment of the risk of the subject patient. The present methods areespecially useful for individuals who have a known genetic risk ofAlzheimer's Disease. Such individuals include those having relatives whohave been diagnosed with the disease, and those whose risk is determinedby analysis of genetic or biochemical markers. Genetic markers of riskfor Alzheimer's Disease include mutations in the APP gene, particularlymutations at position 717 and positions 670 and 671 referred to as theHardy and Swedish mutations respectively. Other markers of risk aremutations in the presenilin genes, PS1 and PS2, and ApoE4, familyhistory of Alzheimer's Disease, hypercholesterolemia or atherosclerosis.Individuals presently suffering from Alzheimer's Disease can berecognized from characteristic dementia, as well as the presence of riskfactors described above. In addition, a number of diagnostic tests areavailable for identifying individuals who have Alzheimer's Disease.These include measurement of CSF tau and Aβ1-42 levels. Individualssuffering from Alzheimer's Disease can also be diagnosed by ADRDAcriteria or the method disclosed herein.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30 years of age). Usually, however, it is not necessary to begintreatment until a patient reaches 40, 50, 60 or 70 years of age.Treatment typically entails multiple dosages over a period of time.Treatment can be monitored by assaying for the presence of ADDLs overtime.

In therapeutic applications, a pharmaceutical composition or medicamentcontaining an antibody or antibody fragment of the invention isadministered to a patient suspected of, or already suffering from such adisease associated with the accumulation of ADDLs in an amountsufficient to cure, or at least partially arrest, the symptoms of thedisease (biochemical, histologic and/or behavioral), including itscomplications and intermediate pathological phenotypes in development ofthe disease. In prophylactic applications, a pharmaceutical compositionor medicament containing an antibody or antibody fragment of theinvention is administered to a patient susceptible to, or otherwise atrisk of, a disease associated with the accumulation of ADDLs in anamount sufficient to achieve passive immunity in the patient therebyeliminating or reducing the risk, lessening the severity, or delayingthe outset of the disease, including biochemical, histologic and/orbehavioral symptoms of the disease, its complications and intermediatepathological phenotypes presenting during development of the disease. Insome methods, administration of agent reduces or eliminates myocognitiveimpairment in patients that have not yet developed characteristicAlzheimer's pathology. In particular embodiments, an effective amount ofan antibody or antibody fragment of the invention is an amount whichachieves at least a 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or97% decrease in the binding of ADDLs to neurons in the patient ascompared to binding of ADDLs in the absence of treatment. As such,impairment of long-term potentiation/memory formation is decreased.

Effective doses of the compositions of the present invention, for thetreatment of the above described conditions vary depending upon manydifferent factors, including means of administration, physiologicalstate of the patient, whether the patient is human or an animal, othermedications administered, and whether treatment is prophylactic ortherapeutic. Usually, the patient is a human but nonhuman mammals suchas dogs or transgenic mammals can also be treated.

Treatment dosages are generally titrated to optimize safety andefficacy. For passive immunization with an antibody or antibodyfragment, dosage ranges from about 0.0001 to 100 mg/kg, and more usually0.01 to 5 mg/kg, of the host body weight are suitable. For example,dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within therange of 1-10 mg/kg. In some methods, two or more antibodies of theinvention with different binding specificities are administeredsimultaneously, in which case the dosage of each antibody administeredfalls within the ranges indicated. Antibodies are usually administeredon multiple occasions, wherein intervals between single dosages can beweekly, monthly or yearly. An exemplary treatment regime entailssubcutaneous dosing, once biweekly or monthly. Advantageously,subcutaneous administration has been found to reduce the flu-likesymptoms associated with intravenous infusions (Lundin, et al. (2002)Blood 100:768-773). Intervals can also be irregular as indicated bymeasuring blood levels of antibody to ADDLs in the patient. In somemethods, dosage is adjusted to achieve a plasma antibody concentrationof 1-1000 μg/mL and in some methods 25-300 μg/mL. Alternatively, theantibody or antibody fragment can be administered as a sustained-releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the antibody inthe patient. In general, human and humanized antibodies have longerhalf-lives than chimeric antibodies and nonhuman antibodies. Asindicated above, dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

Antibody and antibody fragments of the instant invention can beadministered as a component of a pharmaceutical composition ormedicament. Pharmaceutical compositions or medicaments generally containthe active therapeutic agent and a variety of other pharmaceuticallyacceptable components. See Remington: The Science and Practice ofPharmacy, Alfonso R. Gennaro, editor, 20th ed. Lippincott Williams &Wilkins: Philadelphia, Pa., 2000. The preferred form depends on theintended mode of administration and therapeutic application.Pharmaceutical compositions can contain, depending on the formulationdesired, pharmaceutically-acceptable, non-toxic carriers or diluents,which are defined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. Diluents are selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution.

Pharmaceutical compositions can also contain large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such aslatex-functionalized SEPHAROSE™, agarose, cellulose, and the like),polymeric amino acids, amino acid copolymers, and lipid aggregates (suchas oil droplets or liposomes).

Administration of a pharmaceutical composition or medicament of theinvention can be carried out via a variety of routes including, but notlimited to, oral, topical, pulmonary, rectal, subcutaneous, intradermal,intranasal, intracranial, intramuscular, intraocular, or intra-articularinjection, and the like. The most typical route of administration isintravenous followed by subcutaneous, although other routes can beequally effective. Intramuscular injection can also be performed in thearm or leg muscles. In some methods, agents are injected directly into aparticular tissue where deposits have accumulated, for example,intracranial injection. In some embodiments, an antibody or antibodyfragment is injected directly into the cranium. In other embodiments,antibody or antibody fragment is administered as a sustained-releasecomposition or device, such as a MEDIPAD™ device.

For parenteral administration, antibody or antibody fragments of theinvention can be administered as injectable dosages of a solution orsuspension of the substance in a physiologically acceptable diluent witha pharmaceutical carrier that can be a sterile liquid such as water,oils, saline, glycerol, or ethanol. Additionally, auxiliary substances,such as wetting or emulsifying agents, surfactants, pH bufferingsubstances and the like can be present in compositions. Other componentsof pharmaceutical compositions are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil,and mineral oil. In general, glycols such as propylene glycol orpolyethylene glycol are suitable liquid carriers, particularly forinjectable solutions. Antibodies can be administered in the form of adepot injection or implant preparation which can be formulated in such amanner as to permit a sustained-release of the active ingredient.

An exemplary composition contains the instant antibody or antibodyfragment formulated as a sterile, clear liquid at a concentration of atleast 10 mg/ml in isotonic buffered saline (10 mM histidine, 150 mMsodium chloride, 0.01% (w/v) POLYSORBATE 80, pH 6.0). An exemplaryantibody formulation is filled as a single dose, 0.6 ml glass vialsfilled with 3.3 ml of solution per vial. Each vial is stopped with aTEFLON-coated stopper and sealed with an aluminum cap.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced delivery.

For suppositories, binders and carriers include, for example,polyalkylene glycols or triglycerides; such suppositories can be formedfrom mixtures containing the active ingredient in the range of 0.5% to10%, or more desirably 1%-2%.

Oral formulations include excipients, such as pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, and magnesium carbonate. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained-releaseformulations or powders and contain 10% -95% of active ingredient, ormore suitably 25%-70%.

Topical application can result in transdermal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins (see Glenn, et al. (1998) Nature391:851). Co-administration can be achieved by using the components as amixture or as linked molecules obtained by chemical crosslinking orexpression as a fusion protein.

Alternatively, transdermal delivery can be achieved using a skin path orusing transferosomes (Paul, et al. (1995) Eur. J. Immunol. 25:3521-24;Cevc, et al. (1998) Biochem. Biophys. Acta 1368:201-15).

An antibody or antibody fragment of the invention can optionally beadministered in combination with other agents that are at least partlyeffective in treatment of amyloidogenic disease. For example, theinstant antibody can be administered with existing palliative treatmentsfor Alzheimer's Disease, such as acetylcholinesterase inhibitors such asARICEPT™, EXELON™, and REMINYL™ and, the NMDA antagonist, NAMENDA™. Inaddition to these approved treatments, the instant antibody can be usedto provide synergistic/additive benefit for any of several approachescurrently in development for the treatment of Alzheimer's Disease, whichinclude without limitation, inhibitors of Aβ production and aggregation.

Antibody and antibody fragments of the instant invention also findapplication in the identification of therapeutic agents that prevent thebinding of ADDLs to neurons (e.g., a hippocampal cell) therebypreventing downstream events attributed to ADDLs. Such an assay iscarried out by contacting a neuron with ADDLs in the presence of anagent and using an antibody of antibody fragment of the invention todetermine binding of the ADDLs to the neuron in the presence of theagent. As will be appreciated by the skilled artisan, an agent thatblocks binding of ADDLs to a neuron will decrease the amount of ADDLsbound to the neuron as compared to a neuron which has not been contactedwith the agent; an amount which is detectable in an immunoassayemploying an antibody or antibody fragment of the instant invention.Suitable immunoassays for detecting neuronal-bound ADDLs are disclosedherein.

Agents which can be screened using the method provided herein encompassnumerous chemical classes, although typically they are organicmolecules, preferably small organic compounds having a molecular weightof more than 100 and less than about 2,500 daltons. Agents encompassfunctional groups necessary for structural interaction with proteins,particularly hydrogen bonding, and typically include at least an amine,carbonyl, hydroxyl or carboxyl group, preferably at least two of thefunctional chemical groups. The agents often contain cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups. Agents canalso be found among biomolecules including peptides, antibodies,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof. Agents are obtained from awide variety of sources including libraries of natural or syntheticcompounds.

A variety of other reagents such as salts and neutral proteins can beincluded in the screening assays. Also, reagents that otherwise improvethe efficiency of the assay, such as protease inhibitors, nucleaseinhibitors, anti-microbial agents, and the like can be used. The mixtureof components can be added in any order that provides for the requisitebinding.

Agents identified by the screening assay of the present invention willbe beneficial for the treatment of amyloidogenic diseases and/ortauopathies. In addition, it is contemplated that the experimentalsystems used to exemplify these concepts represent research tools forthe evaluation, identification and screening of novel drug targetsassociated with amyloid beta induction of tau phosphorylation.

The present invention also provides methods for detecting ADDLs anddiagnosing a disease associated with accumulation of ADDLs using anantibody or antibody fragment of the instant invention. A diseaseassociated with accumulation of ADDLs is intended to include any diseasewherein the accumulation of ADDLs results in physiological impairment oflong-term potentiation/memory formation. Diseases of this type include,but are not limited to, Alzheimer's Disease and similar memory-relateddisorders.

In accordance with these methods, a sample from a patient is contactedwith an antibody or antibody fragment of the invention and binding ofthe antibody or antibody fragment to the sample is indicative of thepresence of ADDLs in the sample. As used in the context of the presentinvention, a sample is intended to mean any bodily fluid or tissue whichis amenable to analysis using immunoassays. Suitable samples which canbe analyzed in accordance with the methods of the invention include, butare not limited to, biopsy samples and fluid samples of the brain from apatient (e.g., a mammal such as a human). For in vitro purposes (e.g.,in assays monitoring oligomer formation), a sample can be a neuronalcell line or tissue sample. For diagnostic purposes, it is contemplatedthat the sample can be from an individual suspected of having a diseaseassociated with accumulation of ADDLs or from an individual at risk ofhaving a disease associated with accumulation of ADDLs, e.g., anindividual with a family history which predisposes the individual to adisease associated with accumulation of ADDLs.

Detection of binding of the antibody or antibody fragment to ADDLs inthe sample can be carried out using any standard immunoassay (e.g., asdisclosed herein), or alternatively when the antibody fragment is, e.g.,a peptide aptamer, binding can be directly detected by, for example, adetectable marker protein (e.g., β-galactosidase, GFP or luciferase)fused to the aptamer. Subsequently, the presence or absence of theADDL-antibody complex is correlated with the presence or absence,respectively, of ADDLs in the sample and therefore the presence orabsence, respectively, of a disease associated with accumulation ofADDLs. It is contemplated that one or more antibodies or antibodyfragments of the present invention can be used in conjunction withcurrent non-invasive immuno-based imaging techniques to greatly enhancedetection and early diagnosis of a disease associated with accumulationof ADDLs.

To facilitate diagnosis the present invention also pertains to a kit forcontaining an antibody or antibody fragment of the instant invention.The kit includes a container holding one or more antibody or antibodyfragments which recognizes multi-dimensional conformation of ADDLs andinstructions for using the antibody for the purpose of binding to ADDLsto form an antibody-antigen complex and detecting the formation of theantibody-antigen complex such that the presence or absence of theantibody-antigen complex correlates with presence or absence of ADDLs inthe sample. Examples of containers include multiwell plates which allowsimultaneous detection of ADDLs in multiple samples.

The invention is described in greater detail by the followingnon-limiting examples.

EXAMPLE 1 General Materials and Methods

ADDL Preparation. ADDLs in F12 medium (Biosource, Camarillo, Calif.)were prepared from Aβ1-42 in accordance with established methods(Lambert, et al. (2001) supra). Briefly, Aβ1-42 peptide (AmericanPeptide Co., Sunnyvale, Calif. or California Peptide Research, Inc.,Napa, Calif.) was weighed and placed in a glass vial capable of holdinga sufficient quantity of HFIP (1,1,1,3,3,3-hexafluoro-2-propanol) toachieve a peptide concentration of 10 mg/mL. HFIP was added to the drypeptide, the vial was capped and gently swirl to mix, and thepeptide/HFIP solution was stored at room temperature for at least onehour. Aliquots (50 or 100 μL, 0.5 or 1.0 mg, respectively) of peptidesolution was dispensed into a series of 1.5 mL conical centrifuge tubes.The tubes were placed in a SPEEDVAC® overnight to remove the HFIP. Tubescontaining the dried peptide film were capped and stored at −70° C. in asealed container with dessicant.

Prior to use, the Aβ1-42 peptide film was removed from −70° C. storageand allowed to warm to room temperature. Fresh DMSO (44 μL/mg of peptidefilm; 5 mM) was added and the peptide/DMSO mixture was incubated on avortex mixer at the lowest possible speed for ten minutes. F12 media (2mL/mg peptide) was dispensed into each tube of DMSO/peptide and the tubewas capped and mixed by inversion. The 100 μM preparation was stored at2-8° C. for eighteen to twenty four hours. The samples were centrifugedat 14,000×g for ten minutes at 2-8° C. The supernatant was transferredto a fresh tube and stored at 2-8° C. until used.

Biotinylated ADDL preparations (bADDLs) were prepared in the same manneras described above for ADDL preparations using 100% N-terminalbiotinylated amyloid beta peptide (American Peptide Company, Sunnyvale,Calif.).

Monomer Preparation. HFIP dry down preparations of amyloid beta (1-40)peptide (Aβ-40) were prepared as outlined for Aβ(1-42) peptide. Thepeptide film was dissolved in 2 mL of 25 mM borate buffer (pH 8.5) permg of peptide, divided into aliquots, and frozen at −70° C. until used.

Primary Neurons. Primary hippocampal cultures were prepared from frozen,dissociated neonatal rat hippocampal cells (Cambrex, Corp., EastRutherford, N.J.) that were thawed and plated in 96-well COSTAR® platesat a concentration of 20,000 cells per well. The cells were maintainedin NEUROBASAL™ media without L-glutamine (GIBCO-BRL™, Gaithersburg, Md.)and supplemented with B27 (GIBCO-BRL™, Gaithersburg, Md.) for a periodof two weeks and then used for binding studies.

Immunocytochemistry. Immunocytochemistry was performed according toestablished methods (Lambert, et al. (2001) supra), except the secondaryantibodies were conjugated to ALEXAFLUOR® 588 (Molecular Probes, Eugene,Oreg.). Antibodies and ADDLs were preincubated for 1 hour at roomtemperature, at a molar ratio of 1:4 antibody:ADDL before application tothe 21-day hippocampal cell culture. For endogenous ADDLs, human brainprotein (prepared as in Lambert, et al. (2001) supra) was incubated withcells for 1 hour before the cells were washed, fixed, and visualized asabove.

Lightly fixed frozen sections (4% paraformaldehyde at 4° C. for 30 hoursand cryoprotected in 40 μm sucrose) from Alzheimer's Disease and controlhippocampus were incubated with antibody (1:1000 in phosphate-bufferedsaline (PBS)) overnight at 4° C. After removal of antibody, sectionswere washed 3 times with PBS and incubated with secondary antibody atroom temperature. Binding was then visualized with DAB (SIGMA™, St.Louis, Mo.). Sections were then counterstained with hematoxylin,mounted, and imaged on a NIKON® ECLIPSE® E600 light microscope with aSPOT™ INSIGHT™ digital video camera (v. 3.2).

ELISA. Polyclonal anti-ADDLs IgG (M90/1; Bethyl Laboratories, Inc.,Montgomery, Tex.) was plated at 0.25 mg/well on IMMULON™ 3 REMOVAWELL™strips (Dynatech Labs, Chantilly, Va.) for 2 hours at room temperatureand the wells blocked with 2% BSA in TBS. Samples diluted with 1% BSA inF12 were added to the wells, allowed to bind for 2 hours at 4° C., andwashed 3× with BSA/TBS at room temperature. Monoclonal antibodiesdiluted in BSA/TBS were incubated for 90 minutes at room temperature anddetected with a VECTASTAIN® ABC kit to mouse IgG. The HRP label wasvisualized with BIO-RAD® peroxidase substrate and read at 405 nm on aDynex MRX-TC microplate reader.

EXAMPLE 2 Isolation of Mouse Antibody Variable Region Sequences

The cDNAs coding for the variable domains of the 20C2 mouse antibodywere cloned and sequenced following a polymerase chain reaction (PCR)using specially designed primers that hybridize to the 5′-ends of themouse constant regions and to the murine leader sequences upstream ofthe V regions. This ensured that the mouse variable region sequencesobtained were complete and accurate. In short, mRNA was extracted frommouse hybridoma cell lines using the QIAGEN® OLIGOTEX® Direct mRNA MiniKit and subsequently converted to cDNA using a first-strand cDNAsynthesis kit. The cDNA was then used as template in PCR reactions toobtain the antibody variable region sequences.

To obtain the light chain variable region sequence, eleven independentPCR reactions were set up using each of the eleven light chain 5′ PCRprimers (MKV-1 to MKV-11) and the 3′ PCR primer MKC-1 (Table 1).

TABLE 1 SEQ 5′ ID Primer Sequence NO: MKV-1 GAT CTC TAG ATG AAG ATT GCC TGT TAG GCT 11 GTT GGT GCT G MKV-2 GAT CTC TAG  ATG GAG WCA GAC ACA CTC CTG 12 YTA TGG GTG MKV-3 AT CTC TAG ATG AGT GTG CTC ACT CAG GTC 13 CTG GSG TTG MKV-4 GAT CTC TAG ATG AGG RCC CCT GCT CAG WTT 14 YTT GGM WTC TTG MKV-5 GAT CTC TAG ATG GAT TTW CAG GTG CAG ATT 15 WTC AGC TTC MKV-6 GAT CTC TAG ATG AGG TKC YYT GYT SAY CTY 16 CTC TGR GG MKV-7 GAT CTC TAG ATG GGC WTC AAA GAT GGA GTC 17 ACA KWY YCW GG MKV-8 GAT CTC TAG ATG TGG GGA YCT KTT TYC MMT 18 TTT TCA ATG MKV-9 GAT CTC TAG ATG GTR TCC WCA SCT CAG TTC 19 CTT G MKV-10 GAT CTC TAG ATG TAT ATA TGT TTG TTG TCT 20 ATT TCT MKV-11 GAT CTC TAG ATG GAA GCC CCA GCT CAG CTT 21 CTC TTC C SEQ 3′ ID Primer Sequence NO:MKC-1 GAT CGA GCT C AC TGG ATG GTG GGA AGA TGG 22 Underlined and italicsequences denote XbaI and SacI restriction sites, respectively. W = A orT, M = A or C, K = G or T, Y = C or T, and R = A or G.

To obtain the heavy chain variable region sequences twelve independentPCR reactions were set up using each of the twelve heavy chain 5′ PCRprimers (MHV-1 to MHV-12) and the appropriate isotype specific 3′ primer(MHCG-1, MHCG -2A, MHCG-2B, MHCG-3) (Table 2).

TABLE 2 SEQ 5′ ID Primer Sequence NO: MHV-1 GAT CTC TAG ATG AAA TGC AGC TGG GGC ATS 23 TTC TTC MHV-2 GAT CTC TAG ATG GGA TGG AGC TRT ATC ATS 24 YTC TT MHV-3 GAT CTC TAG ATG AAG WTG TGG TTA AAC TGG 25 GTT TTT MHV-4 GAT CTC TAG ATG RAC TTT GGG YTC AGC TTG 26 RTT T MHV-5 GAT CTC TAG ATG GGA CTC CAG GCT TCA ATT 27 TAG TTT TCC TT MHV-6 GAT CTC TAG ATG GCT TGT CYT TRG SGC TRC 28 TCT TCT GC MHV-7 GAT CTC TAG ATG GRA TGG AGC KGG RGT CTT 29 TMT CTT MHV-8 GAT CTC TAG ATG AGA GTG CTG ATT CTT TTG 30 TG MHV-9 GAT CTC TAG ATG GMT TGG GTG TGG AMC TTG 31 CTT ATT CCT G MHV-10 GAT CTC TAG ATG GGC AGA CTT ACC ATT CTC 32 ATT CCT G MHV-11 GAT CTC TAG ATG GAT TTT GGG CTG ATT TTT 33 TTT ATT G MHV-12 GAT CTC TAG ATG ATG GTG TTA AGT CTT CTG 34 TAC CTG SEQ 3′ ID Primer Sequence NO:MHCG-1 GCATC GAG CTC CAG TGG ATA GAC AGA TGG GGG 35 MHCG-GCATC GAG CTC CAG TGG ATA GAC CGA TGG GGG 36 2A MHCG-GCATC GAG CTC CAG TGG ATG AGC TGA TGG GGG 37 2B MHCG-3GCATC GAG CTC CAA GGG ATA GAC AGA TGG GGC 38 Underlined and italicsequences denote XbaI and SacI restriction sites, respectively. W = A orT, M = A or C, K = G or T, Y = C or T,and R = A or G.

Each of the light chain PCR reactions contained 46 μL INVITROGEN™PLATINUM® PCR Super Mix, 1.0 μL of one of the 100 μM 5′ primers (MKV-1to MKV-11), 1.0 μL of the 100 μM 3′ primer (MKC-1), and 2.0 μl ofhybridoma cDNA. Similar PCR reactions were employed to clone the mouseheavy chain variable region sequences. Reactions were placed in a DNAthermal cycler and, after an initial denaturation step at 97° C. for 2.0minutes, subjected to 30 cycles of: 95° C. for 30 seconds, 55° C. for 45seconds, and 72° C. for 90 seconds. Following the last cycle, a finalextension step at 72° C. for 10 minutes was employed. To determine whichPCR reactions yielded product, 5 μL aliquots from each reaction wereseparated on 1.5% (w/v) agarose/1×TAE buffer gels, containing 0.5 μg/mLethidium bromide. PCR products from reactions that produced fragments ofthe expected size (420 to 500 bp) were then gel purified, digested withXbaI and Sad and ligated into the XbaI and Sad sites in the multicloningregion of plasmid pNEB193 (New England Biolabs, Beverly, Mass.).Alternatively, PCR products were ligated directly into plasmid pCR®2.1using the INVITROGEN™ TA CLONING® kit. Ligation products were thentransformed into XL-1 cells and aliquots of the transformed E. coli wereplated onto LB agar plates containing 50 μg/mL ampicillin and overlaidwith 40 μL of X-Gal stock (50 mg/mL) and 40 μl IPTG (100 mM) solutionfor blue/white selection. Plates were incubated overnight at 37° C. andpotential clones were identified as white colonies. DNA from at least 24independent clones for each PCR product were sequenced on both strandsusing universal forward and reverse primers for pNEB193 and pCR®2.1. Theresulting sequences were then assembled into a contig to generate aconsensus sequence for each antibody light and heavy chain variableregion. Using this approach the sequences were determined for the lightand heavy antibody variable regions of hybridoma 20C2 (FIGS. 1A-1B). Thesix complementarity-determining regions (CDRs), which form the structurecomplementary to the antigen, are underlined in FIGS. 1A-1B.

EXAMPLE 3 Humanization of Mouse Anti-ADDL Antibody Variable RegionSequences

Mouse antibody heavy and light variable domain nucleic acids obtainedfrom mouse hybridoma cell line 20C2 were humanized using a CDR graftingapproach. It will be appreciated by those skilled in the art thathumanization of mouse antibody sequences can maximize the therapeuticpotential of an antibody by improving its serum half-life and Fceffector functions thereby reducing the anti-globulin response.

Humanization by CDR grafting was carried out by selecting the humanlight and heavy chain variable regions from the NCBI protein databasewith the highest homology to the mouse variable domains. The mousevariable region sequences were compared to all human variable regionsequences in the database using the protein-protein Basic LocalAlignment Search Tool (BLAST). Subsequently, mouse CDRs were joined tothe human framework regions and the preliminary amino acid sequence wasanalyzed. All differences between the mouse and human sequences in theframework regions were evaluated particularly if they were part of thecanonical sequences for loop structure or were residues located at theVL/VH interface (O'Brien and Jones (2001) In: Antibody Engineering,Kontermann and Dubel (Eds.), Springer Laboratory Manuals). Frameworkregions were also scanned for unusual or rare amino acids in comparisonto the consensus sequences for the human subgroup and for potentialglycosylation sites. Wherein amino acid sequence differences existedbetween the mouse and human framework region sequences that were notfound to be involved in canonical sequences, or located at the VL/VHinterface, the human residue was selected at that position. Wherein adifference in a key residue existed, two versions of the variable regionsequence were generated for evaluation. The CDR grafting strategy madethe minimum number of changes to the human framework region so that goodantigen binding was achieved while maintaining human framework regionsthat closely matched the sequence from a natural human antibody. Theheavy chain and light chain variable region amino acid sequences of theresulting humanized antibody generated by CDR grafting of murine 20C2are shown in FIGS. 2A and 2B, respectively. This antibody is designatedherein as Hu20C2.

Humanized sequences for 20C2 were also designed using a veneeringstrategy (See, e.g., U.S. Pat. No. 6,797,492). Humanization was carriedout by selecting the human light and heavy chain variable regions fromthe NCBI protein database with the highest homology to the mousevariable domains, as well as to the closest human antibody germlinefamily or families (see, Kabat, et al. (1991) 5^(th) ed., Sequences ofproteins of immunological interest, U.S. Dept. Health and HumanServices, NIH, Washington D.C.). The mouse variable region sequenceswere compared to all human variable region sequences in the databaseusing protein-protein BLAST. The murine variable sequences and theirclosest human homologues were modeled to the closest crystallized humanantibody as determined by computer modeling as practiced in the art.From the model of the murine VH and VL sequences, a surface area map wasconstructed, which dictated the solvent accessibility of the amino acidsin the mouse heavy and light variable regions. To confirm the modeling,these exposed residues were compared position-by-position with knownsurface accessible residues (see, e.g., Padlan (1994) Mol. Immunol.31(3):169-217). A score was assigned for each residue in the sequencedesignating it as exposed, mostly exposed, partly buried, mostly buriedand buried according to established methods (see, U.S. Pat. No.6,797,492, incorporated herein by reference in its entirety). Mouseframework residues that scored as exposed or mostly exposed and differedfrom the homologous human sequence were changed to the human residue atthat position. The designed veneered sequences retained the mouse CDRs,residues neighboring the CDRs, residues known be involved in canonicalsequences, residues located at the VL/VH interface, and residues at theN-terminal sequences of the mouse heavy and light chain. The N-terminalsequences are known to be contiguous with the CDR surface and arepotentially involved in ligand binding. Once the veneered sequences werefinalized they were remodeled to look for are any potential obviousstructural issues. A total of 12 and 9 amino acid residues were changedin the heavy chain and light chain frames, respectively. The heavy chainand light chain variable region amino acid sequences of the resultinghumanized antibody generated by veneering of murine 20C2 are shown inFIGS. 2A and 2B, respectively. This antibody is designated herein asHu20C2A3.

In comparison to 20C2, it is noted that the light chain substitutionsresulting in Hu20C2A3, but not the heavy chain substitutions, are incommon with Hu20C2 (FIG. 2). In particular, heavy chain variable regionCDR3 is unique to Hu20C2A3.

Once the humanized amino acid sequences were selected the sequences werereverse-translated to obtain the corresponding DNA sequence. The DNAsequences were codon-optimized using art-established methods (Lathe(1985) J. Mol. Biol. 183(1):1-12) and designed with flanking restrictionenzyme sites for cloning into human antibody expression vectors. Thenucleotide sequences encoding the light chain variable region and twoversions of the heavy chain variable region for Hu20C2 are presented inFIGS. 3A-3C. The two heavy chain variable region versions differ by asingle amino acid substitution at position 24; heavy chain variableregion for version A of Hu20C2 is Phe at position 24 and heavy chainvariable region of version B of Hu20C2 is Leu at position 24.

EXAMPLE 4 Affinity Maturation

Affinity maturation was carried out on the Hu20C2 antibody. Nucleic acidmolecules encoding humanized Hu20C2 versions A and B variable heavychain only, light chain only or heavy chain version A and light chaintogether were cloned in the Fab phage-display vector pFab4. Nucleic acidsequence analysis confirmed sequence and orientation in pFab4. Theannotated Hu20C2 Fab sequences in pFab4 are presented in FIGS. 4A-4C andset forth herein as SEQ ID NO:116 for heavy chain version A, SEQ IDNO:117 for heavy chain version B, and SEQ ID NO:118 for the light chain.The nucleotide sequence for heavy chain version A and light chaintogether in the pFab4 vector is presented in FIGS. 4D-4E. Theseconstructs were used in the Hu20C2 maturation program usingart-established phage-displayed Fab library methods.

Light Chain Maturation. Two libraries were designed to mutate the ninewild-type amino acids of CDR3 of the light (kappa) chain of Hu20C2(i.e., Phe-Gln-Gly-Ser-Leu -Val-Pro-Leu-Thr; SEQ ID NO:39). Theselibraries were designated LC3-1 and LC3-2 representing light chain CDR3sequences of Xaa-Xaa-Xaa-Xaa-Xaa-Val-Pro-Leu-Thr (SEQ ID NO:40) andPhe-Gln-Gly-Ser-Xaa-Xaa-Xaa-Xaa-Xaa (SEQ ID NO:41), respectively.Biotinylated reverse primers, 20C2LC3-1 (SEQ ID NO:123) and 20C2LC3-2(SEQ ID NO:126), were used in combination with forward primer 20C2LC3F(SEQ ID NO:120) to generate the LC3-1 and LC3-2 libraries (see FIG. 5A).Primers were purified by polyacrylamide gel electrophoresis, whereas thevector DNA was purified by gel electrophoresis and electroelution. Thetwo light chain libraries were designed to be randomly mutated. Thefinal diversities of the three 10G5H6 LC₃ libraries were 4.76×10⁸ and7.45×10⁸, respectively (Table 3). Sequence analysis of approximately 100clones from the libraries showed 100% diversity of mutant clones at thedesigned amino acid positions.

TABLE 3 Light Chain Library Characteristic LC3-1 LC3-2 VectorpFab3d20C2HS pFab3d20C2HS Number of 4.76 × 10⁸ 7.45 × 10⁸ TransformantsLibrary Diversity 4.76 × 10⁸ × 0.89 = 7.45 × 10⁸ × 0.90 = 4.24 10⁸ 6.7110⁸ Primary Library 2 mL 2 mL Volume Primary Library 2.13 × 10¹¹ *9.3 ×10¹⁰ Titer *Higher titers are achieved by concentration or phage rescue.

Soluble panning of the two light chain libraries against high molecularweight bADDL was completed. Briefly, four rounds of panning were carriedout using biotinylated high molecular weight ADDL (bADDL). The firstthree rounds were carried out using approximately 1.5 μM antigenconcentration (input=1×10¹⁰ to 1×10¹¹). Upon completion of the thirdround, the outputs of the two libraries were combined and divided intothree groups for analysis with 10 nM, 100 nM and approximately 1.5 μMantigen to increase panning stringency. As such, a total of 58 outputplates were tested in phage ELISA assays, i.e., two plates per libraryin the first round (a total of four plates), six plates per library inthe second round (a total of 12 plates), eight plates for LC₃-1 and 10plates for LC3-2 libraries in the third round (a total of 18 plates) andeight plates for each antigen concentration in the fourth round (a totalof 24 plates).

Panning resulted in 1000 hits, 436 of which were sequenced (Table 4).

TABLE 4 ELISA Round Antigen Input Output % Recovery Screen* Sequenced1^(a)  1.6 μM 2.13 × 10¹⁰ 2.13 × 10⁴ 3.42 × 10⁻⁶   0% 0  (0/176) 2^(a) 2.0 μM 1.55 × 10¹¹ 1.88 × 10⁵ 1.21 × 10⁻⁶ 1.5% 8  (8/528) 3^(a)  1.1 μM1.80 × 10¹⁰  7.8 × 10⁴  4.3 × 10⁻⁶ 5.8% 41  (41/704) 1^(b)  1.6 μM 9.30× 10⁹  5.7 × 10⁴ 6.13 × 10⁻⁶ 2.3% 4  (7/176) 2^(b)  2.0 μM 1.23 × 10¹¹1.07 × 10⁵  8.7 × 10⁻⁷ 4.5% 24  (24/528) 3^(b)  1.1 μM 1.37 × 10¹⁰ 3.32× 10⁵ 2.42 × 10⁻⁵  15% 134 (134/880) 4^(c)  1.1 μM  3.0 × 10¹¹ 1.37 ×10⁵  4.6 × 10⁻⁷  39% — (274/704) 4^(c) 100 nM  3.0 × 10¹¹ 3.88 × 10⁵1.29 × 10⁻⁶  41% — (290/704) 4^(c)  10 nM  3.0 × 10¹¹  1.6 × 10⁵  5.3 ×10⁻⁷  32% 225 (225/704) Total 1000/5104 436 ^(a)20C2 LC3-1 versus highmolecular weight 10% bADDL. ^(b)20C2 LC3-2 versus high molecular weight10% bADDL. ^(c)20C2 LC3-1 + 20C2 LC3-2 versus high molecular weight 10%bADDL. *Hits per total number of colonies.

Sequence and frequency of highly enriched clones are presented in Table5.

TABLE 5 Clone SEQ Desig- ID Round Round Round  nation LC CDR 3 NO: 2 3 4Total Hu20C2LC FQGSLVPLT 39 6 15 14 35 SJ-p1-31 ADTTHVPLT 42 1 2 3SJ-p1-14 AHSTFVPLT 43 1 1 2 4 4P2-12-E3 AQASFVPLT 44 2 2 SJ-p1-38AQATKVPLT 45 1 1 2 4P3-59 AQSSKVPLT 46 2 2 SJ-p2-14 AQSTLVPLT 47 1 2 34P3-11 FAASSVPLT 48 2 2 4P3-1 FESTYVPLT 49 2 2 SJ-p2-10 FESSRVPLT 50 1 12 SJ-p2-11 FNATWVPLT 51 2 2 SJ-p2-60 FQASRVPLT 52 1 5 6 SJ-p1-18FQATRVPLT 53 1 5 6 SJ-p3-51 FQGSFIGLS 54 1 1 2 SJ-p3-16 FQGSFIPGT 55 2 35 SJ-p8-8F FQGSFLPPS 56 1 1 2 SJ-p3-26 FQGSFLPQL 57 1 2 3 SJ-p3-15FQGSLFPPV 58 1 2 3 SJ-p2-70 FQGSLFSPS 59 1 5 6 SJ-p3-24 FQGSRIPIS 60 1 12 SJ-p3-33 FQGSRLPVS 61 2 3 5 SJ-p3-14 FQGSRVPLV 62 2 1 3 SJ-p2-1FFQSSFVPLT 63 6 8 14 4P1-22 FQSSRVPLT 64 15 15 SJ-p2-44 GQTTLVPLT 65 1 34 SJ-p1-56 HESTLVPLT 66 2 1 3 4P1-40 HQSSKVPLT 67 4 4 SJ-p2-20 IQTSLVPLT68 2 2 SJ-p1-41 IQAALVPLT 69 1 1 2 SJ-p2-13 LQSSFVPLT 70 1 4 5 4P1-26LETSRVPLT 71 3 3 SJ-p1-33 LASSHVPLT 72 2 1 3 SJ-p2-27 LNSTTVPLT 73 2 4 6SJ-p2-62 LQSKSVPLT 74 2 2 4P2-26-E5 LQSVRVPLT 75 3 3 4P1-32 LQSSLVPLT 765 5 SJ-p2-37 LQTGRVPLT 77 2 2 4 SJ-p2-64 LQTSFVPLT 78 3 3 4P1-20LQTSNVPLT 79 5 5 SJ-p2-39 LQTTRVPLT 80 2 6 8 SJ-p2-52 LSSTFVPLT 81 3 1 4SJ-p2-6L LSSTHVPLT 82 2 1 3 4P1-77 LTSSAVPLT 83 2 2 SJ-p1-59 LVSSLVPLT84 2 2 SJ-p2-23 METANVPLT 85 2 2 SJ-p1-9M MQSSFVPLT 86 1 3 4 SJ-p2-28MQSSLVPLT 87 1 2 3 SJ-p1-21 MQTSKVPLT 88 1 1 2 4P1-17 SQARMVPLT 89 3 3SJ-p2-66 SQASRVPLT 90 1 2 3 SJ-p1-49 TQSTQVPLT 91 2 1 3 SJ-p2-24VCATFVPLT 92 1 1 2 4P1-41 VQSSAVPLT 93 2 2 SJ-p2-51 VQTSLVPLT 94 12 3143 4P1-64 VQTSVVPLT 95 3 3 SJ-p2-55 VQTTAVPLT 96 2 2 SJ-p1-25 LQTARVPLT97 1 3 4

Fab fragments from the 10 top clones based on enrichment frequency wereprepared and a total of 15 clones were converted into IgG1 humanized Aversion and two clones, 20C2-6 and 20C2-8, were converted to IgG1humanized B version. K_(D) values for these clones were measured byBIACORE™ using biotin-Aβ1-20 (Table 6) and bADDL (Table 7) as antigens.Dramatic improvements in affinity were observed as compared to parentalhumanized 20C2A and 20C2B, as well as mouse 20C2 antibodies. Inparticular, low nanomolar to sub-picomolar K_(D)s were achieved with alight chain CDR3 of the sequence Xaa₁-Gln-Xaa₂-Thr-Arg-Val-Pro -Leu-Thr(SEQ ID NO:2), wherein Xaa₁ is Phe or Leu, and Xaa₁ is Ala or Thr.Moreover, a comparison between K_(D) values obtained with BIACORE™ usingbiotin-Aβ1-20 and bADDL further demonstrates that anti-ADDL antibodiessuch as Hu20C2 preferentially bind multi-dimensional conformations ofADDLs over monomeric Aβ peptides.

TABLE 6 SEQ ID K_(D) (Biotin-Aβ1-20) Name Clone LC-CDR3 NO: Fab IgG1#1IgG1#2 20C2- SJ-p2-60 FQASRVPLT 52  91 nM   1.2 nM — 1A 20C2- SJ-p1-18FQATRVPLT 53  28 nM 686 pM   2 nM 2A 20C2- SJ-p3-16 FQGSFIPGT 55 —  1.7 nM — 3A 20C2- SJ-p2-1F FQSSFVPLT 63  41 nM 912 pM   1.5 nM 5A20C2- 4P1-22 FQSSRVPLT 64  18 nM 544 pM 714 pM 6A 20C2- 4P1-22 FQSSRVPLT64 —  53 pM — 6B 20C2- SJ-p2-27 LNSTTVPLT 73 128 nM — — 7A 20C2-SJ-p2-39 LQTTRVPLT 80  14 nM 140 pM 376 pM 8A 20C2- SJ-p2-39 LQTTRVPLT80 —  46 pM  64 pM 8B 20C2- SJ-p2-51 VQTSLVPLT 94  36 nM 241 pM 420 pM9A 20C2- SJ-p3-33 FQGSRLPVS 61 —  84 nM — 10A 20C2- SJ-p3-6 FQGSLLPLS 98— — — 11A 20C2- 4P1-32 LQSSLVPLT 76 617 nM   1.5 nM — 12A 20C2- 4p1-20LQTSNVPLT 79  94 nM   3 nM — 13A 20C2- SJ-p1-9M MQSSFVPLT 86 126 nM  1.8 nM — 18A 20C2- SJ-p3-15 FQGSLFPPV 58  21 nM 20A 20C2- SJ-p2-66SQASRVPLT 90   2.3 nM 22A 20C2- 4P1-40 HQSSKVPLT 67 649 pM   1.5 nM 23A20C2- SJ-p2-44 GQTTLVPLT 65   1.9 nM 24A 20C2A FQGSLVPLT 39  27 nM 20C2BFQGSLVPLT 39   5.4 nM Mouse- FQGSLVPLT 39  83 nM   3.4 nM 20C2

TABLE 7 SEQ ID K_(D) (bADDL) Name Clone LC-CDR3 NO: Fab IgG1#1 IgG1#220C2- SJ-p2-60 FQASRVPLT 52  85 nM  75 pM — 1A 20C2- SJ-p1-18 FQATRVPLT53  28 nM  15 pM  0.3 pM 2A 20C2- SJ-p3-16 FQGSFIPGT 55 —   3.7 nM — 3A20C2- SJ-p2-1F FQSSFVPLT 63  41 nM 317 pM 68 pM 5A 20C2- 4P1-22FQSSRVPLT 64  42 nM   4.3 pM 24 pM 6A 20C2- 4P1-22 FQSSRVPLT 64 —  53 pM— 6B 20C2- SJ-p2-27 LNSTTVPLT 73 435 nM — — 7A 20C2- SJ-p2-39 LQTTRVPLT80  13 nM   3 pM  0.7 pM 8A 20C2- SJ-p2-39 LQTTRVPLT 80 —  13 pM  0.8 pM8B 20C2- SJ-p2-51 VQTSLVPLT 94  40 nM —  2 pM 9A 20C2- SJ-p3-33FQGSRLPVS 61 —   7.7 nM 10A 20C2- SJ-p3-6 FQGSLLPLS 98 — — — 11A 20C2-4P1-32 LQSSLVPLT 76 238 nM  15 pM — 12A 20C2- 4p1-20 LQTSNVPLT 79 567 nM764 pM 13A 20C2- SJ-p1-9M MQSSFVPLT 86  85 nM 149 pM 18A 20C2- SJ-p3-15FQGSLFPPV 58   6.9 nM 20A 20C2- SJ-p2-66 SQASRVPLT 90 198 pM 22A 20C2-4P1-40 HQSSKVPLT 67  85 pM 66 pM 23A 20C2- SJ-p2-44 GQTTLVPLT 65 114 pM24A 20C2A FQGSLVPLT 39 20C2B FQGSLVPLT 39 Mouse- FQGSLVPLT 39  62 nM  4.1 nM 20C2

Heavy Chain Maturation. The heavy chain of Hu20C2 was also subjected tooptimization by generation of 3 libraries covering the heavy chain-CDR3(RQLGLRSIDAMDY; SEQ ID NO:99). These libraries were designated20C2B-39HC₃-1, 20C2B-39HC₃-2, and 20C2B-39HC₃-3 representing heavy chainCDR3 sequences of XXXXXRSIDAMDY (SEQ ID NO:100) and RQLGLRSIXXXXX (SEQID NO:101) and RQLGXXXXXAMDY (SEQ ID NO:102), respectively. Biotinylatedreverse primers, 20C2HC₃-1 (SEQ ID NO:130), 20C2HC₃-2 (SEQ ID NO:133),and 20C2HC3-3 (SEQ ID NO:136) were used in combination with forwardprimer 20C2HC3F (SEQ ID NO:127) to generate the 3 libraries (see FIG.5B). The libraries contained >10⁸ functional diversity and covered allcombinations of amino acids at every position randomized in each set(see Table 8).

TABLE 8 Monoclonal Antibody Diversity Sequence SEQ ID NO: Hu20C2RQLGLRSIDAMDY 99 20C2B-39HC3-1 5.78 × 10⁸ XXXXXRSIDAMDY 10020C2B-39HC3-2 6.16 × 10⁸ RQLGLRSIXXXXX 101 20C2B-39HC3-3 3.99 × 10⁸RQLGXXXXXAMDY 102

A total of 18 output plates from 4 rounds of panning were tested in aphage ELISA assay. A total of 1235 hits were found, of which 704 weresequenced. Based on BIACORE™ K_(D) values of Fab fragments againstbiotinylated-Aβ1-20 and biotinylated ADDL (bADDL) antigens, as well assingle-point BIACORE™ 3000 analysis (Table 9), a total of 6 Fab cloneswere converted into IgG1 and IgG2m4 using either CDR grafting orveneering humanization techniques. One of these 6 Fabs, designated4a-A3, was isolated from library 20C2B -39HC₃-3 and carried 3 amino acidsubstitutions (RQLGTRGTDAMDY; SEQ ID NO:3) in the middle section of theheavy chain. As is evident from FIG. 2B, this heavy chain CDR3 sequenceis that of Hu20C2A3.

TABLE 9 bADDL Aβ 1-20 BIACORE ™ SEQ Binding Binding 3000 ID CloneK_(D) (M) K_(D) (M) Off-rate Sequence NO: 4a-A3 8.23E−11 8.49E−106.65E−05 RQLGTRGTDAMDY 3 4b-A7 3.76E−10 1.72E−09 1.23E−04 RQLGKLALDAMDY142 4b-H11 1.05E−09 1.33E−09 1.32E−04 RQLGRRSVDAMDY 8 20C28B 1.10E−091.10E−09 8.76E−05 RQLGLRSIDAMDY 143 4a-F5 1.39E−09 1.33E−09 1.17E−04RQLGKLKTDAMDY 7 4a-B2 1.92E−09 1.29E−09 1.13E−04 RQLGARKTDAMDY 5 4b-D82.23E−09 1.69E−09 1.45E−04 RALSPRSIDAMDY 4 4a-A4 2.67E−09 1.58E−091.20E−04 RALSPRSIDAMDY 4 4b-A1 2.87E−09 2.85E−09 1.23E−04 RQLGPRKRDAMDY6 4a-A7 3.24E−09 2.21E−09 1.55E−04 RQLGQRQTDAMDY 144 4a-B9 3.44E−093.54E−09 1.94E−04 RAIQPRSIDAMDY 145 4a-B3 4.17E−09 3.64E−09 1.59E−04RQLGLRSIDAHTR 146 4a-G10 4.52E−09 2.72E−09 1.78E−04 RQLGQPSVDAMDY 1474a-E11 4.93E−09 3.48E−09 1.65E−04 RQLGFQSTDAMDY 148 4a-C9 8.43E−092.46E−09 1.75E−04 RQLGQAGHDAMDY 149 4a-D5 1.17E−09 3.91E−09 1.74E−04RQLGDNVADAMDY 150 4a-E10 1.85E−08 3.60E−09 1.39E−04 RQLGFQSTDAMDY 1484b-D4 1.86E−08 4.87E−09 1.89E−04 RQLGMATPDAMDY 151 4b-B10 6.28E−087.43E−09 1.81E−04 RQLGAHWLDAMDY 152 4b-A12 1.54E−07 1.01E−08 1.69E−04RQLGPEPQDAMDY 153

EXAMPLE 5 Generation of IgG2m4 Antibodies

IgG2m4 antibody derivatives were prepared to decrease Fc receptorengagement, C1q binding, unwanted cytotoxicity or immunocomplexformation while maintaining both the long half-life and pharmacokineticproperties of a typical human antibody. The basic antibody format ofIgG2m4 is that of IgG2, which has been shown to possess a superiorhalf-life in experimental models (Zuckier, et al. (1994) Cancer Suppl.73:794-799). The structure of IgG2 was modified to eliminate C1qbinding, through selective incorporation of IgG4 sequences, whilemaintaining the typical low level of FcγR binding (Canfield and Morrison(1991) J. Exp. Med. 173:1483-1491). This was achieved by usingcross-over points wherein sequences of IgG2 and IgG4 were identical,thereby producing an antibody containing natural Fc sequences ratherthan any artificial mutational sequences. The advantages of using theinstant IgG2m4 antibody which exhibits minimal effector-related activityis comparable to the deglycosylated antibody disclosed by Wilcock et al.((2006) J. Neurosci. 26:5340-6).

The IgG2m4 form of the human antibody constant region was formed byselective incorporation of human IgG4 sequences into a standard humanIgG2 constant region, as shown in FIG. 6. Conceptually, IgG2m4 resultedfrom a pair of chain-swaps within the CH2 domain as shown in FIG. 6.Four single mutations were made corresponding to sequences from IgG4.The Fc residues mutated in IgG2 included His268Gln, Val309Leu,Ala330Ser, and Pro331Ser, which minimized the potential for neoepitopes.The specific IgG4 amino acid residues placed into the IgG2 constantregion are shown in Table 10, along with other alternatives from thebasic structure.

TABLE 10 Residue Residue Alternative (Kabat Residue Residue in residuein numbering) in IgG2 in IgG4 IgG2m4 IgG2m4 Comment 189 Pro or Pro ThrPro Key polymorphism of IgG2; Thr* Pro residue present in IGHG*01allotype and Thr residue present in IGHG2*02 allotype^(a,b). 268 His GlnGln — Change in the B/C loop known to be involved in FcγRII binding^(c).309 Val Leu or Leu Val FcRn binding domain Val 330 Ala Ser Ser — Keyresidue for C1q binding^(d); also potentially involved in binding FcγRIIand FcγRIII^(e). 331 Pro Ser Ser — Key residue for C1q binding^(d,f) andFcγRI binding^(g); also potentially involved in binding FcγRII andFcγRIII^(e). 397 Met or Val Met Val Val residue present in Val* IGHG*01allotype and Met residue present in IGHG2*02 allotype^(a). *Positionsmarked with an asterisk are subject to allelic variations. ^(a)Hougs, etal. (2001) Immunogenetics 52(3-4): 242-8. ^(b)WO 97/11971. ^(c)Medgyesi,et al. (2004) Eur. J. Immunol. 34: 1127-1135. ^(d)Tao, et al. (1991) J.Exp. Med. 173: 1025-1028. ^(e)Armour, et al. (1999) Eur. J. Immunol. 29:2613. ^(f)Xu, et al. (1994) J. Biol. Chem. 269: 3469-3474. ^(g)Canfieldand Morrison (1991) J. Exp. Med. 173: 1483.

Human IgG1/kappa and IgG2m4/kappa versions of humanized Hu20C2 andHu20C2A3 antibodies were constructed. The complete amino acid sequenceof the light and heavy chain Hu20C2A3 IgG2m4 antibody is shown in FIGS.7A and 7C.

EXAMPLE 6 Binding Affinity and Specificity of Humanized Anti-ADDLAntibodies

Affinity maturation was carried out to improve affinity and improvepreferential binding to ADDL. To evaluate ADDL binding affinity of thehumanized antibodies, BIACORE™ and titration ELISAs were conducted asdisclosed herein. Briefly, Streptavidin-coated, 96-well microtiterplates (Sigma, St. Louis, Mo.) were coated with 10% biotinylated ADDLantigen (1 μM). A series of 2-fold dilutions of purified antibody,starting at 500 ng/mL was added to the ADDL captured plates and theplates were incubated for 2 hours at 25° C. After washing five timeswith PBS solution using a plate washer (Bio-Tek, Winooski, Va.),polyclonal goat anti-human kappa light chain antibody (Biomeda, FosterCity, Calif.) was added at a 1/2000 dilution in 3% non-fat milk blockerand incubated at room temperature for 1 hour. A rabbit anti-goat IgG(H+L) HRP-conjugated (Bethyl Laboratories, Inc., Montgomery, Tex.)detection antibody was then added at a 1/2000 dilution in blockingsolution and incubated for 1 hour at room temperature. After washingwith PBS, HRP substrate, 3,3′,5′5-tetramethylbenzidine (ready-to-useTMB; Sigma, St. Louis, Mo.) was added and the reaction was stopped after10 minutes with 0.5 N H₂SO₄. Absorbance at wavelength of 450 nm was readin a plate reader (model VICTOR V; Perkin Elmer, Boston, Mass.) and datawere processed using EXCEL® work sheet. Assay variations between plateswere estimated within 20%.

The K_(D) of Fab clone A3, as measured by BIACORE™, was 849 pM againstbiotinylated-Aβ1-20. The K_(D) of the same Fab clone was 82 pM againstbADDLs, indicating that the A3 Fab demonstrated preferential binding toADDLs. When the clone was humanized by veneering and converted to a fullIgG1 molecule or full IgG2m4 molecule (i.e., Hu20C2A3), the K_(D) valuesagainst Aβ1-20 and ADDL were below the reliable detection limit of theBIACORE™ instrument, indicating a significant improvement of Hu20C2A3'sbinding equilibrium constant against Aβ1-20 and ADDL as compared toHu20C2.

Hu20C2A3, which is the veneered version of clone A3 with an IgG2m4isotype, was expressed in both CHO and Pichia. The two sources ofHu20C2A3 were evaluated for their ability to interact with Aβ monomerand ADDLs by ELISA. As shown in FIG. 8, Hu20C2A3 produced in either CHO(FIG. 8A) or Pichia (FIG. 8B) showed preferential ADDL binding versusAβ40 monomer binding (6-fold). Binding constants (IgGk₅₀ values) asdetermined from these curves yielded values of 64 pM and 376 pM forADDLs and Aβmonomer, respectively for Hu20C2A3 produced in Pichia and 58pM and 361 pM for ADDLs and Aβ monomer, respectively for Hu20C2A3produced in CHO cells.

EXAMPLE 7 Inhibition of ADDL Binding to Neurons Using HumanizedAnti-ADDL Antibodies

The humanized anti-ADDL antibodies were further evaluated for theirability to block ADDL binding to primary hippocampal neurons using themethods disclosed herein. Hu20C2A3 antibody, or PBS as a control, wasmixed at various molar ratios with bADDLs and incubated for one hour at37° C. on a slow rotator. After the preincubation, the antibody/bADDLpreparations were added to primary neuron cultures and incubated for anadditional hour at 37° C. At the end of the incubation period, thebADDLs/antibody mixture was removed and the plates washed six times withmedia. The cells were then fixed in 4% paraformaldehyde for ten minutesat room temperature, the solution removed, fresh fixative added, and thecells fixed for an additional ten minutes. The cells were permeabilizedwith 4% paraformaldehyde containing 0.1% TRITON™ X-100 (2 times, eachfor ten minutes at room temperature), washed six times in PBS and thentreated with 10% BSA in PBS for one hour at 37° C. Alkalinephosphatase-conjugated streptavidin (1:1,500 in 1% BSA; MolecularProbes, Eugene, Oreg.) was then added to the cells for one hour at roomtemperature. The cells were rinsed six times with PBS, the alkalinephosphatase substrate (CDP-STAR® with SAPPHIRE-II™; Applied Biosystems,Foster City, Calif.) added to the cells and incubated for thirty minutesprior to determining the luminescence on a LJL Luminometer (Analyst AD;LJL Biosystems, Sunnyvale, Calif.). In this analysis, Hu20C2A3 was foundto effectively inhibit bADDL binding to neurons at a sub-stoichiometricantibody to peptide ratio (EC₅₀=0.16; FIG. 9).

The inhibition of bADDL binding indicated that Hu20C2A3 interacts withADDLs in a biologically relevant manner. To demonstrate that Hu20C2A3interacts with ADDLs relevant to the human Alzheimer's Diseasecondition, the ability of biotinylated Hu20C2A3 to immuno-label Aβcontaining plaques in human Alzheimer's Disease brain tissue wasevaluated. Immunohistochemical localization showed avid labeling of Aβin both dense core and diffuse plaques in human Alzheimer's Diseasebrain tissue. The specificity of this binding was demonstrated by a lossof immunoreactivity following pre-incubation with increasingADDL:antibody amounts. Similar to Hu20C2, Hu20C2A3, efficiently labelsboth dense core and diffuse Aβ deposits.

EXAMPLE 8 Thermal Stability of Hu20C2A3

An evaluation of the protein stability of Hu20C2A3 was assessed usingSEC-HPLC, fluorescent thermal melt analysis, and particle size analysis.Fluorescent thermal melt analysis indicated that Fc and Fab unfoldingtransitions occurred at approximately 70° C. and 80° C., respectively,consistent with acceptable inherent protein stability (FIG. 10).

EXAMPLE 9 In Vivo Pharmacodynamic and Efficacy Analysis

The prior art indicates that systemic injection of monoclonal anti-Aβantibodies can increase plasma levels of Aβ acutely, whereas measurablelowering of brain Aβrequires chronic administration. It has beensuggested that passive immunization in species with measurable Aβresults in an elevation of plasma Aβ due to a change in the equilibriumof Aβ between brain and peripheral compartments. This “peripheral sink”ultimately leads to lowering of brain Aβ. However, cognitive improvementhas been observed in animals following acute antibody administrationprior to notable changes in brain Aβ, indicating that changes in brainAβ may occur in some form prior to a time point where these changes canbe measured using known techniques. Alternately, elevations of plasma Aβcould be explained by a stabilization of peripheral Aβ followingadministration of antibody. Regardless of the interpretation, it hasbeen established that early plasma elevations are a prerequisite forsubsequent lowering of brain Aβ in animal models. Thus, the effect ofHu20C2A3 antibodies on plasma Aβ elevations were utilized as anindicator of target engagement. Following infusion of Hu20C2A3 at dosesof 30, 100 and 300 μg/mouse IV, significant and robust increases inplasma Aβx-40 were observed relative to the non-relevant antibody (8B4)control group 4 hours post-injection (FIG. 11). The observed increasesin plasma Aβx-40 for CHO-derived material were 491% (30 μg, p>0.001),826% (100 μg, p<0.001), and 755% (300 μg, p<0.001) of the 8B4 levels.Similarly, the increases in plasma Aβx-40 for Pichia-derived materialwere 395% (30 μg, p>0.001), 729% (100 μg, p<0.001), and 838% (300 μg,p<0.001) of the 8B4 levels.

1. An isolated antibody, or an antigen binding fragment of the antibody,that binds amyloid β-derived diffusible ligands comprising: (a) a lightchain variable region comprising, (i) a CDR1 of SEQ ID NO:154, (ii) aCDR2 of SEQ ID NO:155, and (iii) a CDR3 of SEQ ID NO:80; and (b) a heavychain variable region comprising, (i) a CDR1 of SEQ ID NO:156, (ii) aCDR2 of SEQ ID NO:157, and (iii) a CDR3 of SEQ ID NO:3.
 2. The isolatedantibody of claim 1, further comprising a heavy chain constant region ofSEQ ID NO:137.
 3. The isolated antibody of claim 1, wherein the antibodyis a monoclonal antibody.
 4. A pharmaceutical composition comprising theantibody or antigen binding fragment of claim 1 in admixture with apharmaceutically acceptable carrier.
 5. A kit for detecting Aβ-deriveddiffusible ligands comprising the antibody or antigen binding fragmentof claim
 1. 6. A method for attenuating binding of Aβ-derived diffusibleligands to a neuron comprising contacting the neuron with the antibodyor antigen binding fragment of claim 1 so that binding of Aβ-deriveddiffusible ligands to the neuron is attenuated.
 7. A method forinhibiting assembly of Aβ-derived diffusible ligands comprisingcontacting a sample containing amyloid β 1-42 peptides with the antibodyor antigen binding fragment of claim 1 thereby inhibiting assembly of Aβ-derived diffusible ligands.
 8. A method for inhibiting thephosphorylation of tau protein at Ser202/Thr205 comprising contacting asample containing a tau protein with the antibody or antigen bindingfragment of claim 1 thereby inhibiting the phosphorylation of tauprotein at Ser202/Thr205.
 9. A method for attenuating the symptoms of adisease associated with Aβ-derived diffusible ligands comprisingadministering an effective amount of the pharmaceutical composition ofclaim
 4. 10. A method for identifying a putative therapeutic agent thatattenuates the binding of Aβ-derived diffusible ligands to neuronscomprising (a) contacting a composition comprising a neuron withAβ-derived diffusible ligands in the presence of an agent; (b)contacting the composition with the antibody or antigen binding fragmentof claim 1; and (c) detecting the amount of antibody or antigen bindingfragment bound to the neuron in the presence of the agent, wherein adecrease in the amount of antibody or antigen binding fragment bound inthe presence of the agent as compared to the amount of antibody bound inthe absence of the agent indicates that the agent is a putativetherapeutic agent for attenuating binding of Aβ-derived diffusibleligands to neurons.
 11. A method for detecting Aβ-derived diffusibleligands in a sample comprising contacting the sample with the antibodyor antigen binding fragment of claim 1 and determining the presence of acomplex comprising the Aβ-derived diffusible ligands and antibody orantigen binding fragment.
 12. A method for diagnosing a diseaseassociated with Aβ-derived diffusible ligands comprising contacting abiological sample with the antibody or antigen binding fragment of claim1 and determining the presence of a complex comprising the Aβ-deriveddiffusible ligands and the antibody or antigen binding fragment whereinthe presence of the complex is diagnostic of a disease associated withAβ-derived diffusible ligands.